RESEARCH LETTER

Mobile elements and mitochondrial genome expansion in the

soil fungus and potato pathogen Rhizoctonia solani AG-3
Liliana Losada1, Suman B. Pakala1, Natalie D. Fedorova1,2, Vinita Joardar1, Svetlana A. Shabalina2,
Jessica Hostetler1,2, Suchitra M. Pakala1, Nikhat Zafar1, Elizabeth Thomas3, Marianela
Rodriguez-Carres3, Ralph Dean3, Rytas Vilgalys4, William C. Nierman1 & Marc A. Cubeta3
1
The J. Craig Venter Institute, Rockville, MD, USA; 2National Center for Biotechnology Information, National Library of Medicine, National
Institutes of Health, Bethesda, MD, USA; 3North Carolina State University, Raleigh, NC, USA; and 4Duke University, Durham, NC, USA

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Correspondence: Marc A. Cubeta, Abstract
Department of Plant Pathology, North
Carolina State University, 851 Main Campus The soil fungus Rhizoctonia solani is an economically important pathogen of
Drive, Raleigh, NC, 27606 USA. agricultural and forestry crops. Here, we present the complete sequence and
Tel.: 919 513 1227; analysis of the mitochondrial genome of R. solani, field isolate Rhs1AP. The
fax: 919 513 0024; genome (235 849 bp) is the largest mitochondrial genome of a filamentous
e-mail: [email protected]
fungus sequenced to date and exhibits a rich accumulation of introns, novel
repeat sequences, homing endonuclease genes, and hypothetical genes. Stable
Received 25 October 2013; revised 24
December 2013; accepted 20 January 2014.
secondary structures exhibited by repeat sequences suggest that they comprise
Final version published online 17 February functional, possibly catalytic RNA elements. RNA-Seq expression profiling con-
2014 firmed that the majority of homing endonuclease genes and hypothetical genes
are transcriptionally active. Comparative analysis suggests that the mitochon-
DOI: 10.1111/1574-6968.12387 drial genome of R. solani is an example of a dynamic history of expansion in
filamentous fungi.
Editor: Holger Deising
MICROBIOLOGY LETTERS

Keywords
Basidiomycota; homing endonucleases;
repetitive elements.

ncbi.nlm.nih.gov/genomes/ORGANELLES/organelles.
Introduction
html). The mt genome size disparity among fungi sug-
The soil fungus Rhizoctonia solani (teleomorph = gests a dynamic history of expansion and contraction,
Thanatephorus cucumeris, Phylum Basidiomycota) is an similar to plant mitochondria (Scott & Logan, 2007).
economically important pathogen of agricultural and for- Several major factors that contribute to mt genome
estry crops. The fungus is also a competitive saprobe and diversity are known and include proliferation of non-
an endomycorrhizal symbiont of orchids that represents coding content (e.g. short dispersed repeats), expan-
an evolutionary link between beneficial and disease-caus- sion of existing introns, gene duplication followed by
ing fungi (Cubeta & Vilgalys, 2000; Rodriguez-Carres inactivation (e.g. partial copies), and acquisition of
et al., 2011). Recently, as a consortium, we initiated a col- homing endonuclease genes (HEGs) or other sequences
laborative project to sequence the complete nuclear and from diverse sources (Sloan et al., 2012). The relatively
mitochondrial (mt) genomes of the potato pathogen small size and mostly uniparental inheritance of fungal
R. solani anastomosis group 3 (AG-3), strain Rhs1AP. mitochondria makes them ideal candidates for study-
Here, we present the assembly, annotation of the R. solani ing evolution, fungicide insensitivity, population
mt genome, and phylogenetic analysis of mt genomes of genetics, and taxonomy (Bullerwell & Lang, 2005; Alv-
related fungi in the Basidiomycota. erson et al., 2010; Joardar et al., 2012). However, these
Fungal mtDNA genomes display diversity in size ranging studies are hampered by the paucity of sequenced mt
from 24 874 bp for Cryptococcus neoformans to 135 005 bp genomes of fungi in the Basidiomycota as less than
for Agaricus bisporus (Ferandon et al., 2013) (http://www. twenty are currently available. Comparative genomic

FEMS Microbiol Lett 352 (2014) 165–173 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
166 L. Losada et al.

analysis suggests that R. solani has the largest Subsequently, mRNA was fragmented into smaller pieces
sequenced mt genome in fungi which is driven by the and reverse-transcribed to cDNA with the aid of Super-
multiplication of novel repetitive elements. The phylo- Script Double-Stranded cDNA Synthesis Kit (Life Tech-
genetic analysis confirmed the position of nologies). After each preparation step, the sample quality
R. solani as an early diverging and transitional lineage and quantity was assessed using Agilent 2100 Bioanalyzer
of the Agaricomycotina. and the Agilent RNA 6000 Nano Kit (Agilent). The cDNA
library construction involved end-repair, A-tailing, adap-
ter ligation, and library amplification followed by cluster
Materials and methods
generation and sequencing. The cDNA library was
sequenced using the Illumina Genome Analyzer II (GA
DNA extraction
II) instrument (www.illumina.com).
Five to seven days mycelial mats of R. solani Rhs1AP

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grown in potato dextrose broth at 24 °C were washed
RNA-Seq data analysis
three times with sterile deionized water and freeze-dried.
Lyophilized tissue was ground into a fine powder in Raw sequence data were processed, filtered, and normal-
liquid nitrogen with a mortar and pestle and used for ized by the Illumina pipeline to generate FASTQ files,
DNA extraction with SDS buffer and phenol chloroform which were analyzed using the RNA-Seq module of CLC
as previously described (Murray & Thompson, 1980; Genomics Workbench. All reads were mapped to
Zolan & Pukkila, 1986; Vilgalys & Gonzalez, 1990). R. solani mt coding sequences to calculate expression val-
Extracted DNA was placed into 10-mL tubes and sub- ues for each gene in RPKM (reads per kilobase exon
jected to cesium chloride ultracentrifugation in a VTi80 model per million mapped reads) units (Mortazavi et al.,
rotor for 12 h at 508 735 g at 20 °C using the procedure 2008). Genes were considered expressed if they had more
of Sambrook et al. (1989). than four sequence reads mapped to their exonic regions.

Sanger sequencing Mitochondrial genome annotation

Sequencing was performed as part of the complete genome The open reading frames (ORFs) were identified using
sequencing project using the Sanger 3730xl sequencing Artemis (Rutherford et al., 2000) with genetic code 4.
instrument to obtain 272 974 reads from libraries with dif- Functional assignments were made based on sequence sim-
ferent insert sizes (4 and 10 kb plasmid, and 40 kb fosmid) ilarity to characterized fungal mitochondrial proteins using
to obtain 28X sequence coverage and linkage of contigs. BLASTP searches against NCBI databases and HMM searches
There was continuous clone coverage which provided clear against the protein families in the Pfam database (Finn
evidence for the circularity of the mt genome. et al., 2010). ORFs that had no significant similarity to
known genes and were at least 65 amino acids in length
were annotated as hypothetical proteins. The tRNAs and
Mitochondrial genome assembly and closure
ribosomal RNAs were identified using tRNAscan-SE (Lowe
The reads were initially assembled with CELERA ASSEMBLER & Eddy, 1997) and Rfam (Griffiths-Jones et al., 2005).
(CA) software version 5.3 (Miller et al., 2008). To assemble
the mitochondrial genome, identified mitochondrial
Repeat identification and analysis
sequences from R. solani and other fungi in the Basidiomy-
cota were used as queries in BLASTN searches against all CA Interspersed repeats were identified using RepeatMasker
output files. The contigs were manually assembled into the (Tarailo-Graovac & Chen, 2009) and a curated fungal-
final genomic sequence, which was trimmed and rotated specific repeat database (Repbase) (Jurka et al., 2005), as
using the finishing package Consed (Gordon et al., 1998). well as with RepeatScout (Price et al., 2005) and
PrintRepeats (http://www.genome.ou.edu/miropeats.html)
using default settings. PSI-BLAST-based sequence homology
RNA sample preparation and Illumina
searches were performed using TransposonPSI. MITE-HUN-
sequencing (RNA-Seq)
TER (Han & Wessler, 2010) was used to search for minia-
Total RNA was isolated from the mycelium of R. solani ture inverted repeat transposable elements (MITEs). The
harvested at 12, 48, 144 and 196 h during the sclerotia nine consensus repeat sequences generated by Repeat-
formation process using TRIzol reagent (Life Technolo- Scout were used as the reference repeat library for Re-
gies). Oligo (dT)-based enrichment for mRNA was car- peatMasker. Outputs from PrintRepeats were clustered
ried out using MicroPoly(A)PuristTM Kit (Ambion). using Usearch (http://www.drive5.com/usearch/), and

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 352 (2014) 165–173
Published by John Wiley & Sons Ltd. All rights reserved
Rhizoctonia solani AG-3 mitochondrial genome 167

BLASTN searches were performed on these clusters and on boot-strapped trees were generated and used to assign the
the nine consensus sequences from RepeatScout. Tandem bootstrap support values to the best-scoring ML tree.
repeats were identified using the program TRF with The JTT amino acid substitution model was used with
default parameters. the Gamma model of rate heterogeneity. Podospora
anserina was used as the outgroup taxon. A maximum
parsimony-based tree was also generated using the PROT-
Phylogenetic analysis
PARS program of the PHYLIP package (Felsenstein, 1989).
Fourteen core proteins (atp6, atp8, atp9, cox1, cox2, cox3,
cob, nadh1, nadh2, nadh3, nadh4, nadh4l, nadh5, and
Results
nadh6) encoded by 13 genomes were concatenated and
aligned using Muscle (Edgar, 2004). Regions with poor
Mitochondrial genome sequence
alignments were removed with Gblocks using default set-

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tings (Castresana, 2000). Maximum-Likelihood (ML) The mt genome of R. solani Rhs1AP is circular, with
trees were generated using the Randomized Axelerated 235 849 bp, and represents the largest fungal mt gen-
Maximum-Likelihood (RAxML) program (Stamatakis ome sequenced to date (Fig. 1a). This size is consistent
et al., 2005). Multiple ML trees were generated, and the with the results of Jian et al., (1998) that estimated a
best-scoring tree was identified. One hundred genome size of c. 200 kb based on restriction analysis

(a) (b)

(c)

Fig. 1. (a) Rhizoctonia solani circular mitochondrial genome. From out to in, track 1 (blue) is the chromosome. The first nucleotide was defined
as the start of the ribosomal operon. Mid-length and long repeats are presented offset as additional units each with its own color (fuchsia,
green, yellow, orange, and dark red). Track 2, all annotated protein-coding elements (bright green) and ribosomal RNAs (brown); protein-coding
ORFs within RNA are colored in dark brown. Repeat elements are colored according to track 1. Track 3 hypothetical and homing endonucleases
are highlighted in orange. Track 4 exons for core genes, including ribosomal RNAs are colored in blue. Track 5 RNA-Seq read coverage (> 4
reads) at each position is highlighted in red. Links: Each connecting line shows the presence of identical sequences at each position. Gray links
represent short repeats (< 35 bp) found up to 100 times in the genome; colored links show the location of the mid- and long repeats and follow
the coloration in track 1. (b) Structure of the COX1 gene showing COX1 exons in yellow connected by lines, other ORFs as yellow arrows and
repeats as blue arrow. Genomic read coverage is shown in pink. (c) Synteny between AG1-IB, AG1-IA, and Rhs1AP mt genomes. Darker lines
represent > 95% identity. The apparent rearrangement is due to different arbitrary nucleotide position 1 for each circular molecule.

FEMS Microbiol Lett 352 (2014) 165–173 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
168 L. Losada et al.

and with the R. solani AG1-IB mitochondrial genome

Abundance of homing endonuclease genes and
recently published (162 751 bp) (Wibberg et al., 2012).
accessory genes
The next largest mt genomes are A. bisporus
(135 005 bp) (Ferandon et al., 2013) and Chaetomium We identified 33 genes encoding homing endonucleases,
thermophilum (127 206 bp), respectively. The mt which are associated with self-splicing mobile genetic
genome of R. solani Rhs1AP had a G + C content of elements. All R. solani Rhs1AP endonucleases belonged
35.9%, with no strand bias. We found no evidence for to the two classes most frequently identified in fungal
heterozygosity in the mt genome, suggesting that it was mitochondria and characterized by the sequence motifs
completely homozygous. In contrast, population genetics LAGLIDADG and GIY-YIG. These motifs are usually
studies of field isolates of R. solani AG-3 showed that found in group I intronic sequences and intergenic
the nuclear genomes are heterozygous (Ceresini et al., regions (Belfort et al., 2005). In total, the genome con-
2007). Unlike other anastomosis groups of R. solani, the tained eight GIY-YIG and 25 LAGLIDADG HEG genes.

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Rhs1AP mt genome had no apparent integrated or free- Twenty-two of these 33 endonucleases were located
replicating linear or circular plasmids (Miyasaka et al., inside introns, and the remaining 11 were present in
1990). Syntenic analysis with two R. solani mitochondria intergenic regions of the genome. Eight core genes con-
showed high identity (> 95%) and general conservation tained introns, five of which contained HEGs. Homing
of core mitochondrial gene sequences and order, but endonucleases are common in fungal and other eukary-
differed significantly in accessory and noncoding regions otic mt genomes (Belfort et al., 2005) and can bind to
(Fig. 1c). asymmetric recognition sequences of 12–40 bp. HEGs
Rhizoctonia solani Rhs1AP mt genome contained 138 are hypothesized to represent ‘semi-selfish elements’ that
genes including 15 core genes found in all Basidiomycota mt promote transfer (homing) of introns by cleaving DNA
genomes. Core genes encoded the hydrophobic subunits of at a specific site (Belfort et al., 2005). Endonucleases can
respiratory chain complexes – atp6, atp8, atp9, cox1, cox2, also function as intron-specific maturases by facilitating
cox3, cob, nadh1, nadh2, nadh3, nadh4, nadh4l, nadh5, (but not catalyzing) RNA splicing (Lambowitz & Perl-
nadh6, and rps3 encoding the 40S ribosomal protein S3. man, 1990; Lambowitz & Belfort, 1993). Introns associ-
Accessory genes include 33 homing endonucleases and 86 ated with mitochondrial genomes are self-splicing due to
hypothetical genes. Additionally, partial copies of atp6, cox2, limited pre-RNA processing in the organelle, and hom-
nadh3, and nadh6 genes were also identified. The partial ing endonucleases can facilitate their splicing and inte-
genes lacked the N-terminus and were located close to their gration into new sites. Thus, acquisition of endonuclease
full length copies (Fig. 1a). The genome encoded 27 tRNAs genes by self-splicing introns in R. solani likely facilitated
for all 20 amino acids and the small and large ribosomal their expansion by converting them into invasive mobile
RNA subunits. All core genes were present on the positive elements (Haugen & Bhattacharya, 2004). In addition to
strand, whereas one tRNA and 20 hypothetical genes were HEGs, the R. solani mt genome contained 86 hypotheti-
found on the negative strand. cal proteins, which have no significant similarity to
Six core genes, cox1, cox2, cox3, nadh1, nadh5, and cob, other protein sequences deposited in public databases,
were intron-rich and contained complex nested intronic PFAM domains, or to each other. The size of the hypo-
structures composed of hypothetical genes, repeats, and thetical genes ranged from 66 to 467 amino acids.
homing endonuclease genes (HEGs) each containing mul- Twenty of the 86 hypothetical genes were found on the
tiple introns. Although known group IB and IIB1 introns negative strand, which suggests that they are of recent
were found in the COX1 and SSU genes (Supporting origin.
Information, Fig. S1), the majority of introns did not
align well with known group I or group II introns. For
One-third of the genome is occupied by
example, the cox1 gene spanned 20 892 bp and had a
interspersed repeats
coding sequence length of 1587 bp in seven exons
(Fig. 1b). Intronic regions in cox1 had 44 repeat elements, The most salient feature of the R. solani Rhs1AP mt gen-
five HEGs, and four hypothetical genes. The fourth intron ome was the presence of interspersed repeats that occupy
contained a LAGLIDADG HEG, which itself had three 34% of the genome. There were at least three types of
repeat-containing introns. Interestingly, the cox1 locus in repeats found in our study: short interspersed palin-
other fungi [e.g. A. bisporus (29 902 bp)] (Ferandon dromic sequences < 40 bp, mid-length (50–95 bp)
et al., 2010) was larger, but the expansion was driven by sequences, and longer elements (> 100–963 bp). No
group I introns and not by repeat elements as observed known transposable or interspersed elements were
in R. solani. detected. The palindromic sequences were each repeated

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 352 (2014) 165–173
Published by John Wiley & Sons Ltd. All rights reserved
Rhizoctonia solani AG-3 mitochondrial genome 169

numerous times, up to 100 times in one case (Fig. 1a). analysis of other fungal genomes in the Basidiomycota
The RNA products of three highly similar mid-length showed no similar repeat structures (Table S1), suggesting
repeated elements were found to have significantly lower that this phenomenon is unique to R. solani and that
folding free energy than those of randomly shuffled their acquisition and evolution warrants further
sequences of the same dinucleotide and nucleotide com- investigation.
position (t-test, P < E-30). Furthermore, these elements
were predicted to fold into conserved and highly stable
Rhizoctonia solani represents basal and
secondary structures that resemble structures of RNAs
transitional lineage within the Agaricomycotina
with enzymatic activity (Fig. 2a). In aggregate, these ele-
ments were repeated 26 times in the genome and were all To understand the evolutionary relationships between
preceded by a candidate promoter sequence R. solani and other fungi in the Basidiomycota, we per-
(TTAATTCGCCTA[A/T]) (Kubelik et al., 1990). One formed a phylogenetic analysis of mt DNA sequences

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additional element found three times had conserved sec- from 14 core proteins (Fig. 3). The coding regions of
ondary structure similar to those found in introns with both R. solani mitochondria were highly similar (> 97%
ribozyme activity (Fig. 2b) and may represent a highly identity). At the time of our analysis, eleven publicly
stable core structure with catalytic activity. Based on our available Basidiomycota mt genomes were included in the
analysis, none of the elements showed all the necessary analysis and a fungus in the Ascomycota, P. anserina, was
characteristics to be classified as MITEs, short inter- used as the out-group taxon. Our results are congruent
spersed elements (SINEs), or other well-characterized with previous findings based on multilocus sequence typ-
classes of TEs, although some had terminal inverted ing analyses (Rodriguez-Carres et al., 2011) and suggest
repeats and others had apparent target site duplications that R. solani represents an early diverging, transitional
(TSD). The presence of TSD provides evidence of past lineage within Agaricomycotina. Earlier studies indicate
transposition events. However, as none of the predicted that R. solani belongs to the Cantharelloid clade in the
genes shared sequence similarity with known transposases Agaricomycotina (mushroom-forming fungi) (Moncalvo
or reverse transcriptases, it is unlikely that any of these et al., 2006). Numerous phylogenies of the Kingdom
elements behave as transposons. Usually, three or more of Fungi (Matheny, 2005; James et al., 2006; Hibbett et al.,
these repeat elements appeared clustered in islands in the 2007) based on analysis of different loci (RPB2, RPB2,
mt genome (Fig. 1a), with the largest (963 bp) containing EF1-alpha, and ribosomal DNA) have shown that differ-
all other elements, except for two of the palindromic ent genera (Botryobasidium, Cantharellus, Ceratobasidium,
repeat elements. These findings suggest two possibilities: Thanatephorus, and Uthatobasidium) in the Cantharelloid
These regions represent hot-spots for insertion, and many clade usually displayed a basal position within the Agaric-
elements jump consecutively into these locations, or a omycotina.
single large element was introduced in the genome, then
copied – and in some cases degenerated. Subsequently,
Majority of accessory genes are
smaller elements were copied around the genome by
transcriptionally active
recombination or possibly transposition using an
unknown transposase as evidenced by TSD. None of the To validate predicted endonuclease and hypothetical
repeat elements were shared between the R. solani genes, RNA samples collected from R. solani were
Rhs1AP and AG1-IB mitochondria, underscoring the rela- sequenced with RNA-Seq technology (Nagalakshmi et al.,
tively large divergence between these groups. Further, our 2008). A total of 114 195 reads mapped to the genes of

(a) (b)

Fig. 2. Predicted secondary structure of the

Rhizoctonia solani repeat elements (a) RSOL-
mtRPT5, highly expressed and preceded by a
candidate promoter, (b) RSOL-mtRPT6, similar
0 1 0 1
to introns with ribozyme activity. Residues are
0 1 0 1
colored according to base pairing probabilities.

FEMS Microbiol Lett 352 (2014) 165–173 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
170 L. Losada et al.

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Fig. 3. Phylogenetic analysis of 14
concatenated core proteins of Cryptococcus
neoformans, Moniliophthora periciosa,
M. roreri, Phakopsora meibomiae,
P. pachyrhizi, Pleurotus ostreatus, Podospora
anserina, Rhizoctonia solani AG-3 isolate
Rhs1AP, R. solani AG1-IB isolate 7/3/14,
Schizophyllum commune, TIlletia indica,
T. walkeri, and Ustilago maydis. Protein
sequences were aligned using MUSCLE and
ML trees were generated and boot-strapped
using the RAxML program as described in
Materials and methods.

the mt genome, of which 31 281 mapped to the protein- functional core set of genes, the genome is comprised of
coding genes while 82 914 mapped to ribosomal RNA a rich collection of introns, homing endonucleases, hypo-
genes and an additional 46 760 to intergenic sequences thetical genes, and novel repetitive elements. Our data
(30% of reads; Fig. S1). The fraction of mapped reads provide evidence for mitochondrial size expansion, which
was lower than anticipated based on the estimated mt to highlights the role of interspersed repeat elements in the
nuclear genome size ratio (0.0024) (M.A. Cubeta and evolution of fungal mitochondria. Although the genetic
D.C. Schwartz, unpublished) and expected mt copy num- mechanisms associated with this expansion are not
ber. However, the data show that many accessory genes known and beyond the scope of this study, there are sev-
were transcriptionally active (Fig. 1a). Eighty of the 138 eral plausible explanations for this observation.
genes were expressed (minimum coverage of four reads) The fungal strain Rhs1AP used in this study repre-
with a median coverage of 72 reads. In addition to the 15 sents a naturally occurring heterokaryotic (N + N)
core genes, 53 hypothetical genes, 6 LAGLIDADG and 3 organism that possesses at least two distinct haploid
GIY-YIG endonuclease genes were also expressed. A nuclear genomes based on whole genome sequence data
hypothetical gene (343.t000094) had the highest expres- and an optical map of the chromosomes (M.A. Cubeta,
sion level among the mt genes. It is noteworthy that W.C. Nierman, and D.C. Schwartz, data not shown).
many of the represented intergenic regions were repeats Information from the optical mapping studies has pro-
surrounding HEG genes or were also present in the vided evidence for recombination and occurrence of at
assembled nuclear genome, suggesting that their relatively least three copies of five different chromosomes that are
high abundance may be in part due to sequence over- distributed across the genome. This genome complexity
abundance rather than to a particular biological function. suggests that our strain has been involved in previous
hyphal fusion (anastomosis) and interaction events that
have contributed to nuclear complexity and possibly the
Discussion
observed cytoplasmic mtDNA diversity. In nature, het-
To our knowledge, the soil fungus and potato pathogen erokaryons of R. solani form as a result of the interac-
R. solani represents the largest mitochondrial genome tion and fusion of hyphae from two compatible haploid
sequenced in a filamentous fungus. In addition to the (homokaryotic) strains during the mating process (Cubeta

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 352 (2014) 165–173
Published by John Wiley & Sons Ltd. All rights reserved
Rhizoctonia solani AG-3 mitochondrial genome 171

& Vilgalys, 1997). However, heterokaryons can also death response of the interacting hyphal cells. It has been
form during the interaction of heterokaryotic and hypothesized that somatic recognition systems have
closely related homokaryotic strains. In both of these evolved in fungi to prevent the introgression of mitochon-
types of hyphal interactions, there is mixing of cyto- drial and nuclear DNA, mycoviruses, plasmids, and other
plasm (heteroplasmy) and mitochondrial DNA. selfish elements and organelles, although alternate hypoth-
Although it is widely accepted that the inheritance of eses related to kin selection have also been proposed (Aa-
mitochondrial DNA in animals and fungi is uniparen- nen et al., 2008). In R. solani, the occurrence of
tal and nonrecombining (Taylor et al., 1986; Griffiths, mitochondrial- and nuclear-associated plasmids and dou-
1996; Basse, 2010), there is also evidence for biparen- ble-stranded RNA (dsRNA) elements are common and
tal and recombining behavior in these organisms can often bypass cell death responses (Tavantzis, 1994;
under laboratory and field conditions (Barr et al., Charlton & Cubeta, 2007) and systems that regulate uni-
2005). For example, Saville et al. (1998) and Smith parental inheritance. Perhaps plasmids and dsRNAs may

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et al. (1990) demonstrated the occurrence of a low promote the spread of associated mitochondrial elements
frequency of mtDNA recombination in a natural pop- that contribute to the diversity and size expansion in the
ulation of the root-infecting basidiomycete fungus mtDNA genome of R. solani observed in this study.
Armillaria gallica. de la Bastide and Horgen (2003) The soil fungus R. solani represents an important basal
also provided experimental evidence for the detection and transitional lineage of filamentous fungi, and the mt
of nonparental mitochondrial DNA haplotypes in genome sequence of this organism will provide a valu-
crosses of homokaryons of the common button able resource to further examine the phylogenetic relat-
mushroom fungus A. bisporus. More recently, Xu edness of mushroom-forming fungi and their allies,
et al. (2000) and van Diepeningen et al. (2010) which have been poorly sampled. The complex nested
reported mtDNA recombination in natural population intronic structures in core genes found in the mt gen-
of the basidiomycete human pathogenic yeast C. gattii ome of R. solani represent common targets of antifungal
and dung-inhabiting filamentous ascomycete fungus agents used to manage plant disease caused by the fun-
P. anserina, respectively. Interestingly, the authors in gus. Interestingly, resistance (insensitivity) in R. solani
the latter study have suggested that mitochondrial anastomosis group 1-IA, a major pathogen that causes
fusion coupled with the activity of homing endonu- sheath blight disease of rice, to the quinone outside
clease genes, which were found in the assembled inhibitor (QoI) strobilurin family of fungicides has been
nuclear genome and adjacent to intergenic regions of recently reported (Castroagudin et al., 2013; Olaya et al.,
the mtDNA in R. solani, may possibly contribute to 2013). Resistance to this fungicide is associated with the
recombination and patterns of mtDNA inheritance. F129L mutation (phenylalanine to leucine substitution at
Recent research also suggests an association of mating codon 129) in the cytochrome oxidase b gene of the
genes and uniparental inheritance of mtDNA in the mtDNA (Olaya et al., 2013). The information generated
human and plant pathogenic basidiomycete fungi in this study will provide a unique opportunity to
C. neoformans and Ustilago maydis (Basse, 2010). Per- understand fungicide resistance mechanisms, and to
haps the nuclear condition (homokaryon vs. hetero- develop diagnostic assays and fungicide resistance moni-
karyon) of the vegetative hyphae of the interacting toring strategies for a plant disease-causing organism of
fungal strain can influence diversity and inheritance global economic importance.
of mtDNA as suggested by Skosireva et al. (2010) that
showed deviations from expected patterns of unipa-
Data availability
rental mtDNA inheritance in crosses of haploid and
nonhaploid strains of C. neoformans. Sequence data for this project has been deposited at
In addition to genetic compatibility systems that regu- DDBF/EMBL/GenBank (BioProject ID: PRJNA73133;
late the fusion of hyphae and mating of fungi, most accession No. KC352446). The mitochondrial genome of
fungi investigated possess somatic recognition systems the button mushroom fungus A. brunnescens (= Agaricus
and an ability to recognize self versus nonself (Glass bisporus) has been recently sequenced and is 172 566 bp
et al., 2004). However, the genetic basis for somatic rec- long (http://genome.jgi-psf.org/Agabi_varbisH97_2/Aga-
ognition systems are not well understood in Rhizoctonia bi_varbisH97_2.home.html).
fungi, but is thought to involve the interaction of multi-
ple loci. In filamentous basidiomycete fungi, somatic
Acknowledgments
incompatibility often occurs between two interacting het-
erokaryotic strains that differ at the loci that regulate We thank Dana Busam, Karen Beeson, Sana Scherbakova,
somatic recognition, which results in a programmed cell and Lakshmi Viswanathan from JCVI for assistance with

FEMS Microbiol Lett 352 (2014) 165–173 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
172 L. Losada et al.

sample processing, library construction, and sequencing. Edgar RC (2004) MUSCLE: multiple sequence alignment with
We are also grateful to Paul Paukstelis from the Univer- high accuracy and high throughput. Nucleic Acids Res 32:
sity of Maryland for helpful suggestions regarding the 1792–1797.
analysis of the RSOL-mtRPT secondary structure. We Felsenstein J (1989) PHYLIP—phylogeny inference package
thank JoAnne Crouch, Frank Martin, and Stellos Tavanti- (Version 3.2). Cladistics 5: 164–166.
zis for presubmission review of this manuscript. Funding Ferandon C, Moukha S, Callac P, Benedetto J-P, Castroviejo
M & Barroso G (2010) The Agaricus bisporus cox1 gene: the
for this study has been provided by a grant from NSF/
longest mitochondrial gene and the largest reservoir of
USDA-CSREES Microbial Genome Sequencing Program
mitochondrial group I introns. PLoS One 5: e14048.
#2007-35600-18550 to W.N., R.D., and M.C. Ferandon C, Xu J & Barroso G (2013) The 135 kbp
mitochondrial genome of Agaricus bisporus is the largest
Authors’ contribution known eukaryotic reservoir of group I introns and
plasmid-related sequences. Fungal Genet Biol 55: 85–91.

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L.L. and S.B.P. contributed equally to this work. Finn RD, Mistry J, Tate J et al. (2010) The Pfam protein
families database. Nucleic Acids Res 38: D211–D222.
Glass NL, Rasmussen C, Roca MG & Read ND (2004) Hyphal
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FEMS Microbiol Lett 352 (2014) 165–173 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved